Cell-surface receptors mediate a variety of biological effects through binding their cognate ligands. Receptors are typically composed of one or more integral membrane proteins that bind the ligand, such as a cytokine or counter-receptor, with high affinity and transduce this binding event to the cell through the cytoplasmic portions of the certain receptor subunits. Cell-surface receptors have been grouped into several classes on the basis of similarities in their extracellular ligand binding domains.
The physiological importance of cell-surface receptors is exemplified by the role of co-stimulatory receptors in mediating immune cell function. T cells are normally activated by T cell antigen receptor (TCR) engagement by MHC molecules plus foreign peptides presented by antigen presenting cells (APCs). Professional APCs also express a number of co-stimulatory molecules that engage other receptors on the T cells and contribute to activation. A proven means of inhibiting T cell activation is interference with engagement of these co-stimulatory molecules. Most notably, CTLA4-Ig can be used to prevent the critical co-stimulatory signals through the CD28 molecule, thereby interfering with T cell responses.
Other co-stimulatory molecules have been identified and these tend to be important in specialized situations where their counter-structures are expressed. One such molecule whose contribution has only recently been appreciated is the co-stimulatory receptor CD226. The counter-structures for CD226 are the nectin-family proteins PVR (CD155) and Nectin-2 (CD112), which are widely expressed in tissues typically infiltrated by lymphocytes in auto-immune or auto-inflammatory diseases including epithelium, endothelium, synoviocytes, and cells of the CNS. Importantly, signals through CD226 have been shown to be critical to T cell activation in situations where T cells are being stimulated by non-professional APC's (see Gilfillan et al., J. Exp. Med. 205:2965-2973, 2008; Iguchi-Manaka et al., J. Exp. Med. 205:2959-2964, 2008), which would include autoimmune diseases where T cells are being activated and causing damage in inflamed tissues. Moreover, a polymorphism in CD226 has recently been linked to risk of developing a number of autoimmune conditions including multiple sclerosis (MS), type I diabetes, Grave's disease, and Wegener's granulomatosis. (See International Multiple Sclerosis Genetics Consortium (IMSGC), Genes Immun. 10:11-14, 2009; Hafler et al., Genes Immun. 10:5-10, 2009; Maier and Hafler, Immunol. Rev. 229:322-336, 2009; Mait et al., “Non-synonymous variant (Gly307Ser) in CD226 is associated with susceptibility to multiple autoimmune diseases,” Rheumatology (Oxford) (Mar. 24, 2010 [Epub ahead of print]).) Hence, there is sound rationale for interfering with CD226-mediated signals in immune cell function.
VSTM3 (also referred to as B7R1 in International PCT Publication No. WO 06/124667) is an inhibitory member of the CD28 family that has been shown to also bind to PVR and Nectin-2, the same counter-structures as CD226. In fact, VSTM3 and CD226 cross-compete for binding to PVR and Nectin-2, indicating that they bind overlapping if not identical regions, although VSTM3 seems to bind with somewhat higher affinity.
Soluble forms of many cell-surface receptors are known. These soluble receptors correspond to the ligand-binding domains of their cell-surface counterparts. For example, naturally occurring soluble cytokine receptors inhibit cytokine responses and act as transport proteins. (See, e.g., Aggarwal and Puri, “Common and Uncommon Features of Cytokines and Cytokine Receptors: An Overview,” in Aggarwal and Puri, eds., Human Cytokines: Their Role in Disease and Therapy, Blackwell Science, 1995, 3-24.) In addition, it has been found that dimerization of soluble receptor polypeptides through the use of fusion proteins may enhance the binding properties of these soluble receptors so that they become therapeutically useful antagonists of their cognate ligands. Typical of such dimeric fusions are immunoglobulin fusions. (See, e.g., Sledziewski et al., U.S. Pat. Nos. 5,155,027 and 5,567,584; Jacobs et al., U.S. Pat. No. 5,605,690; Wallner et al., U.S. Pat. No. 5,914,111; and Ashkenazi and Chamow, Curr. Opin. Immunol. 9:195-200, 1997.)
For example, soluble VSTM3 has been shown to be therapeutically efficacious in animal models of T-cell-mediated disease. In particular, it has been shown that soluble VSTM3-Fc dimers, in a conventional bivalent format, inhibit T cell responses in vivo, as measured by a Delayed Type Hypersensitivity (DTH) reaction, and that such dimers decrease disease incidence and progression in a Collagen Induced Arthritis (CIA) model. (See International PCT Publication No. WO 06/124667.) A soluble VSTM3-VASP tetramer has also been shown to be efficacious in the CIA model. (See id.)
A variety of biological effects, including the growth and differentiation of many cell types, are also mediated by small, soluble proteins collectively referred to as cytokines (see, e.g., Arai et al., Annu. Rev. Biochem. 59:783, 1990; Mosmann, Curr. Opin. Immunol. 3:311, 1991; Paul and Seder, Cell 76:241, 1994). Proteins that constitute the cytokine group include interleukins, interferons, colony stimulating factors, tumor necrosis factors, and other regulatory molecules.
The demonstrated in vivo activities of cell-surface receptors and cytokines illustrate the clinical potential of, and need for, molecules that mediate the biological activities of receptors and cytokines. For example, demonstrated in vivo activities of pro-inflammatory receptors and cytokines, including the activities of co-stimulatory receptors in mediating immune cell responses, illustrate the enormous clinical potential of, and need for, antagonists of pro-inflammatory molecules. There is particularly a need for soluble fusions of such cell-surface receptors and cytokines having improved activity relative to known fusion protein formats. The present invention, as set forth herein, meets these and other needs.